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Originally published In Press as doi:10.1074/jbc.M611551200 on April 25, 2007

J. Biol. Chem., Vol. 282, Issue 26, 19103-19112, June 29, 2007
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Capsular Arabinomannans from Mycobacterium avium with Morphotype-specific Structural Differences but Identical Biological Activity*Formula

Manon Wittkowski{ddagger}, Jessica Mittelstädt§, Sven Brandau§1, Norbert Reiling, Buko Lindner||, Jordi Torrelles**23, Patrick J. Brennan**3, and Otto Holst{ddagger}4

From the {ddagger}Structural Biochemistry, §Immunotherapy, Molecular Infection Biology, and ||Biophysics, Leibniz-Center for Medicine and Biosciences, Research Center Borstel, D-23845 Borstel, Germany and the **Department of Microbiology, Immunology, and Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, Colorado 80523-1682

Received for publication, December 18, 2006 , and in revised form, April 10, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The capsules of two colony morphotypes of Mycobacterium avium strain 2151 were investigated, i.e. the virulent smooth-transparent (SmT1) and the nonvirulent smooth-opaque (SmO) types. From both morphotypes we separated a nonacylated arabinomannan (AM) from an acylated polysaccharide fraction by affinity chromatography, of which the AMs were structurally characterized. The AMs from the virulent morphotype, in contrast to that from the nonvirulent form, possessed a larger mannan chain and a shorter arabinan chain. Incubation of murine bone marrow-derived macrophages and human dendritic cells showed that the acylated polysaccharide fractions were potent inducers of tumor necrosis factor-{alpha}, interleukin-12, and interleukin-10 compared with nonacylated AMs, which led to only a marginal cytokine release. Further in vitro experiments showed that both the acylated polysaccharide fractions and the nonacylated AMs were able to induce in vitro anti-tumor cytotoxicity of human peripheral blood mononuclear cells. Thus, morphotype-specific structural differences in the capsular AMs of M. avium do not correlate with biological activity; however, their acylation is a prerequisite for effective stimulation of murine macrophages and human dendritic cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Numerous bacteria, in particular extraintestinal and invasive pathogens, express capsules that enable them to decorate surface molecules, leading to inhibition of phagocytosis. Other encapsulated intracellular pathogens protect themselves against phagocytic digestion by actively killing macrophages.

Some mycobacterial species contain capsular-like outer layers, which consist mainly of polysaccharides, proteins, and small amounts of lipids (1). First evidence of the presence of a mycobacterial capsule was provided in 1959 by Chapman et al. (2) as a "capsular space" between the phagosomal membrane of the infected cell and a mycobacterium. Electronmicroscopy led to the term "electron transparent zone." In 1986 the existence of the capsule was confirmed by chemical analyses (3). Unlike other bacterial capsules, the mycobacterial capsule does not seem to offer protection against phagocytosis. In contrast, mycobacteria have developed strategies to penetrate into macrophages, which usually are responsible for initial nonspecific degradation of pathogens but which, during innate immunity, present mycobacteria the optimal environmental conditions for survival and replication (4). Like other capsules, those of mycobacteria consist mainly of polysaccharides. The function of such capsular polysaccharides has not been completely determined up to now. They may protect the bacteria against enzymatic digestion after phagocytosis. Furthermore, capsular polysaccharides possibly trigger the interaction between bacteria and host cells during the first steps of infection, and it has been assumed that they contribute to intracellular persistence and replication. Taken together, capsular polysaccharides are believed to be potent virulence factors.

Three polysaccharides of the capsule of Mycobacterium tuberculosis have been isolated and partly characterized, i.e. a glycogen-like glucan with an estimated molecular mass of 100 kDa, a mannan, and an arabinomannan (AM)5 (1). The exact localization and arrangement of these polysaccharides remain to be determined. Both glucan and AM were expressed in vivo and in vitro (5, 6). It has been shown by immunoelectron microscopy that the AM is localized in the capsule (7). Another modified AM possessing serological activity has been isolated from Mycobacterium smegmatis (8).

The exact structure of the AM (estimated molecular mass, 13 kDa) from M. tuberculosis has not been fully characterized. The proposed structure comprises a mannan backbone substituted by a branched arabinan, which is further modified by Man residues at the nonreducing end. The arabinan is structurally similar to the arabinan of the arabinogalactan located in the cell wall (9, 10).

Mycobacterial surface molecules including lipoarabinomannan (LAM) and AMs have been shown to stimulate and activate immune cells. These immunomodulatory properties have ultimately led to the use of such compounds in vaccination against mycobacterial infections and as immunotherapeutic agents in cancer therapy (11-19). AMs preparations show profound anti-tumor activity in murine experimental cancer models, and mechanistic studies reveal that the anti-tumor activity of AMs depends on distinct immune cell populations and TH1 cytokines (16, 17). However, these reported studies were performed on Z-100, an extract from M. tuberculosis strain Aoyama B, which was described as containing AMs as well as LAM. Thus, it remains unclear whether acylation plays a role in the identified biological activities. Thus far, little is known about the structural characteristics and the potential biological role of AMs from other mycobacterial species.

Infection with Mycobacterium avium, a facultative intracellular opportunistic pathogen in humans, is a well studied model for mycobacterial infections. A major phenotypic characteristic of M. avium isolates is their ability to appear in different colony morphotypes, smooth (either transparent or opaque) or rough (reviewed in Ref. 20). Although morphotypes of the same strain are very closely related, previous studies have demonstrated that the smooth-transparent variant is usually better able to survive and grow intracellularly than the opaque morphotype (21, 22). With regard to the M. avium morphotypes used in this study (2151 SmT and SmO), this pattern has been confirmed both in murine macrophages and in a mouse model of infection (23, 24).

In the current study we purified AMs of SmT and SmO of M. avium strain 2151. To correlate the structural properties with biological activity we used cytokine production by human and murine dendritic cells and induced cytotoxicity of human peripheral blood mononuclear cells (MNCs) as read-out systems. As a result we found distinct structural differences in the AM polysaccharide composition of SmT compared with that of SmO, which did not correspond to differences in biological activity. Our studies also revealed that AMs from M. avium could induce cytotoxicity of human immune effector cells in vitro.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Chemical Reagents—All chemicals were of highest grade. Aqueous solutions for chemical reactions were prepared with Milli-Q® water.

Bacteria and Growth Conditions—Mycobacterium avium, serovar 2, strain 2151, was originally isolated from a patient with AIDS (25). The colony morphotypes SmT and SmO were grown in stable form on Middlebrook 7H10-agar (19.0 g liter-1, Difco), containing nutrient broth (4.0 g liter-1, Difco), glycerol (8.0 g liter-1, Merck), glucose (5.5 g liter-1, Merck), sodium pyruvate (0.76 g liter-1, Merck), and sodium glutamate (0.5 g liter-1, Merck). The pH was adjusted to 7.7. Cultivations comprised 150 agar plates (150 x 20 mm, Sarstedt, with 100 ml medium each) and proceeded at 37 °C for 7 weeks. Yields were 0.5-1.0 g wet mass/plate.

Isolation of Polysaccharides—Aqueous phenol (1%, w/v) was added to the freshly harvested wet cells (18 ml g-1), and the suspensions were gently shaken at 4 °C for 48 h. After centrifugation at 27,000 x g at 4 °C for 20 min, the supernatant was filtrated (0.22 µm, Steritop, Millipore), dialyzed against MilliQ-H2O (5 x 10 liters) at 4 °C, using 3,500 MWCO membrane (SpectraPor, Roth) to remove phenol and low-molecular mass components, and finally lyophilized (yield: 1.5% of the bacterial wet mass). This capsular material was extracted with 10% aqueous Triton X-114 (Sigma, St. Louis, MO) at 37 °C as described (26) to eliminate lipoglycans (i.e. phosphatidylinositol mannosides) and lipoproteins. After phase separation, the aqueous layer was precipitated with cold acetone, and the obtained sediment was washed once with cold acetone and then dried under a stream of nitrogen (0.26% of the bacterial wet mass). The aqueous phase was resuspended in a small volume of water and added to 46% (w/v) ammonium sulfate saturation, and incubated at 20-22 °C for 4 h, then at 4 °C for 18 h, to precipitate the proteins. The sample was centrifuged (14,000 x g, 4 °C, 60 min), yielding a precipitate composed mostly of proteins and a supernatant containing mostly polysaccharides. To remove the salt the sediment and the supernatant were extensively dialyzed at 4 °C against aqua bidest.(6 x 10 liters) and freeze-dried. To remove rests of the protein the polysaccharide-rich supernatants were incubated with Proteinase K (10 µgml-1; Boehringer, 50 °C, 5 h), dialyzed and finally freeze-dried.

Size Exclusion Chromatography—Proteinase K-treated polysaccharide-rich fractions were applied on a Sephadex G-50 column (3 x 80 cm; Amersham Biosciences) at 20-22 °C and eluted with pyridine/acetic acid/water (4/10/1000 by volume) at a flow rate of 0.5 ml min-1. The void volume was determined using dextran blue (10 mg ml-1; molecular mass, 2 x 106 Da). Separated fractions were detected by a differential refractometer, combined, concentrated on a rotary evaporator, and freeze-dried.

High Performance Liquid Affinity Chromatography—Separations were performed utilizing a preparative high performance liquid chromatography equipment (HPLC, Abimed-Gilson) at 20-22 °C and a lectin-Sepharose column (ConA-Sepharose 4B, 5 x 1 cm, Sigma) at a flow rate of 0.8 ml min-1. The following gradient was applied: 0.1 M sodium acetate, 0.1 M NaCl, 1 mM MgCl2, 1 mM MnCl2, 1 mM CaCl2, pH 6.1, for 40 min and then 0.5 M {alpha}-methyl mannopyranoside in the above eluent for 20 min. UV absorbance was detected at 210 and 280 nm. Obtained fractions were dialyzed extensively against aqua bidest. at 4 °C and then freeze-dried.

SDS-PAGE and Lectin Blotting—SDS-PAGE was performed using a MiniProtean 3 cell (Bio-Rad) at 200 V for 50 min. Gels (8 x 7 x 1 mm) were prepared with 12% acrylamide for the separating gel and 5% acrylamide for the stacking gel and were stained by Coomassie Brilliant Blue R-250 (Bio-Rad) (27) and periodic acid Schiff reagent (Merck) (28). Electroblotting onto polyvinylidene difluoride membranes (Immobilon-P, Millipore) was performed by using a Mini Trans-blot electrophoresis transfer cell (Bio-Rad) at 100 V for 1 h. Blotted membranes were washed three times in Tris-HCl-buffered saline/TBST (20 mM Tris, 150 mM NaCl, 0.1 mM MnCl2, 0.1 mM CaCl2, 0.1% Tween 20, pH 7.7) and then incubated with 20 ml of TBST containing 100 µg of ConA (Sigma) at 20-22 °C for 1 h. After washing five times with H2O, blots were developed by a reaction mixture of 7 ml of 4-chloro-1-naphthol (Sigma, 3 mg ml-1 methanol), 2 ml of 0.2 M Tris-HCl (pH 7.7), and 20 µl of 30% H2O2 (Sigma) in the dark for 15 min. After washing once with H2O, blots were dried.

Analyses—For protein quantification a bicinchoninic acid (BCA) microassay was performed according to the supplier's (Pierce) instructions.

The endotoxin contents of fractions A and B isolated by affinity chromatography were established by a chromogenic Limulus amoebocyte lysate assay according to the manufacturer's instructions (Coamatic Chromo-LAL).

Sugar composition and methylation analyses (methylation was repeated twice) were performed as described (29). Separations were performed on a Hewlett-Packard gas chromatograph 438A equipped with a flame ionization detector and a poly-(5% diphenyl-95% dimethyl)-siloxane SPB-5 capillary column (inner diameter, 30 m x 0.32 mm; film thickness, 0.25 µm; Supelco). Hydrogen was used as a carrier gas at 60 kilopascals. Alditol acetates were separated at 150 °C for 3 min and then, increasing linearly, at 3 °C min-1 to 300 °C.

Mass Spectrometry—Identification of partially methylated alditol acetates was performed by GC/MS on a Hewlett-Packard mass spectrometer (HP5989A) equipped with a HP-5MS capillary column. Helium was used as carrier gas at a pressure of 10 p.s.i. The initial temperature of 150 °C was increased after 3 min with 5 °C min-1 to a final temperature of 320 °C. Electron impact mass spectra were recorded at 70 eV. For chemical ionization, NH3 was used as the reactant gas.

Isolated AMs were analyzed by high resolution electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI FT-ICR-MS). Measurements were performed on a 7-Tesla-APEX II (Bruker Daltonics) equipped with an Apollo ion source. Spectra were recorded in the positive-ion mode. Samples were dissolved in a mixture of 2-propanol, water, and 30 mM ammonia acetate, pH 4.70 (50:50:0.03, v/v/v), in concentrations of 10 µg µl-1 and sprayed with a flow rate of 2 µl min-1. In experiments using capillary skimmer dissociation the capillary exit voltage was set to 250 V. Theoretical masses of polysaccharide fragments were calculated according to the formula: mass = (a·mAra) + (b·mMan) + mH2O + (c·mNa) - (c·H) atomic mass units.

NMR Spectroscopy—NMR experiments were performed on a Bruker DRX AVANCE 600 spectrometer operating at 600.31 MHz for 1H and 150.96 MHz for 13C. Samples were repeatedly exchanged with D2O (500 µl, 99.9% purity, Deutero) with intermediate freeze-drying and then dissolved in 500 µl of D2O (99.99% purity, Deutero). All one- and two-dimensional spectra were recorded at 27 °C. Chemical shifts were expressed in ppm and relative to internal acetone ({delta}H 2.225; {delta}C 31.45). COSY, TOCSY, NOESY, and ROESY spectra were measured using data sets (t1 x t2) of 512 x 2048 points. TOCSY and NOESY experiments were carried out in the phase-sensitive mode with mixing times of 100 and 200 ms, respectively. Heteronuclear two-dimensional 1H,13C correlation was measured by HMQC and HMBC experiments with data sets of 256 x 2048 points. For homo- and heteronuclear correlations experiments, 128 scans were acquired.

Isolation and Stimulation of Murine Bone Marrow Macrophages—Bone marrow-derived macrophages were isolated from 8-10-week-old C57BL/6 mice (Charles River) as described (30); 5 x105 macrophages (106 ml-1) were stimulated with the isolated polysaccharide fractions A and B in concentrations of 1 and 10 µgml-1 at 37 °C, 5% CO2 for 24 h. Supernatants from stimulated murine macrophages were examined for the presence of TNF-{alpha}. Detection was performed with an anti-mouse TNF-{alpha} kit according to the manufacturer's instructions (DuoSet mouse TNF-{alpha}/TNFSF1A, R&D Systems).

Generation of Human Monocyte-derived DC—Peripheral blood mononuclear cells were isolated from citrate blood of healthy donors by discontinuous gradient centrifugation (Biocoll (separating solution), Biochrom). Monocytes were elutriated by counterflow centrifugation and concentrated to 106 cells ml-1 RPMI 1640 medium containing 5% fetal calf serum (Linaris), 100 units ml-1 penicillin, and 100 µgml-1 streptomycin. Differentiation into immature DC was achieved by a 7-day culture in the presence of granulocyte/macrophage-stimulating factor (500 units ml-1) and IL-4 (500 units ml-1).

106 DC ml-1 were stimulated with 10 µgml-1 isolated polysaccharide fractions A and B at 37 °C, 5% CO2 for 18 h. The supernatants were examined for the presence of TNF-{alpha}, IL-10, and IL-12 (Ready-Set-Go, human IL-12 ELISA, eBiosciences) by using enzyme-linked immunosorbent assay kits according to the manufacturer's instructions.

Stimulation of Human MNC—Human MNC were stimulated with polysaccharide fractions A and B in concentrations of 10 and 40 µgml-1, Mycobacterium bovis BCG (Connaught substrain, Immucyst ®) with an multiplicity of infection of 0.04 cell-1 and phosphate-buffered saline (100 µl) were added. Cells were cultured for 7 days in 6-well microtiter plates at 37 °C, 5% CO2.

Cell Culture—The human bladder tumor cell line T-24 was cultivated at 37 °C, 5% CO2 in RPMI 1640 (PAA Laboratories) containing 10% fetal calf serum (Linaris), 1% glutamate, 100 units ml-1 penicillin, and 100 µgml-1 streptomycin.

Chromium Release Assay—Cytotoxicity of stimulated human MNC against tumor cells was determined in a standard chromium release assay. Target cells were harvested, washed, and labeled with 50 µCi of Na 512CrO4 (Hartmann)/106 cells for 1.5 h at 37 °C. After washing, the cells were resuspended with a final concentration of 5 x 104 cells ml-1. Effector cells were harvested and added to a total of 100 µl of target cells at effector/target ratios of 40:1, 20:1, and 10:1.

The radioactive content of the supernatant was measured in a {gamma}-counter (Berthold) after incubation for 5 h at 37 °C,5%CO2. The specific lysis was determined according to the formula: specific lysis (%) = (Reff - Rspo)/(Rmax - Rspo) x 100. By adding 100 µl of medium, the spontaneous release of radioactivity (Rspo) was defined. The maximum release (Rmax) was achieved by adding 100 µl of lysis solution. The experimental release of radioactivity, which was induced by the effector cells (Reff), was measured in triplicates.


Figure 1
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  		FIGURE 1.
Separation of fraction 2 after Sephadex G-50 from SmO using high-performance liquid affinity chromatography. Separations were performed on a lectin-Sepharose column (ConA-Sepharose 4B, Sigma; 5 x 1 cm). Eluent A, 0.1 M sodium acetate, 0.1 M NaCl, 1 mM MgCl2, 1 mM MnCl2, 1 mM CaCl2, pH 6.1. Eluent B, A + 1 M {alpha}-methyl mannopyranoside. At a flow rate of 0.8 ml min-1 the following gradient was used: 100% A for 40 min, 100% B for 20 min, 100% A for 60 min. UV absorbance was measured at 210 nm (···) and 280 nm (- - -). The volume of each fraction was 0.8 ml.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Extraction and Purification of AM—Applying a mild aqueous phenol extraction method (31), capsular material was extracted from the envelope of M. avium. SDS-PAGE of this phenol extract showed numerous proteins after Coomassie staining. Successive blotting and staining with ConA-conjugated peroxidase revealed the occurrence of LAM, lipomannan, and phosphoinositolmannan (not shown). The lipoglycans not only are membrane-associated (32) but also are thought to be present in the outer surface of the bacilli (33) and thus extractable under mild conditions. Routine monosaccharide analysis showed the presence of sugars in the phenol extract, namely Ara, Man, Glc, Gal, and two different O-methylhexoses. To separate the lipoglycans, an additional extraction utilizing the detergent Triton X-114 was performed (33). The resulting detergent phase included LAM, lipomannan, and phosphoinositolmannan, and the aqueous phase comprised proteins and carbohydrates. Precipitation with ammonium sulfate separated proteins from carbohydrates. The protein-containing sediments are presently being investigated separately. The supernatants (containing carbohydrates) were further fractionated by size exclusion chromatography on Sephadex G-50. Three base-line-separated fractions were obtained, the last of which was identified as salt. Fraction 1 (eluting in the void volume) contained Glc and Gal in an approximate molar ratio of 1:3. Fraction 2 comprised Ara and Man in an approximate molar ratio of 5:4 (preparation of M. avium 2151, SmO) and 10:7 (preparation of SmT), as well as Glc and O-methylhexose in minor amounts. Fractions 2 of both SmO and SmT were separated on ConA-Sepharose, from which ConA-nonreactive acylated polysaccharides (fraction A) and ConA-reactive arabinomannans (fraction B) were isolated (Fig. 1).

Compositional Analyses—The same sugars were identified in fractions A from SmO and SmT, however, in different ratios. Arabinose was identified in both samples in similar concentrations (SmO, 502 nmol mg-1; SmT, 477 nmol mg-1). The major component was a 6-O-methyl-hexose (SmO, 1069 nmol mg-1; SmT, 779 nmol mg-1). Another 3-O-methyl-hexose was less abundant in fraction A from SmO (126 nmol mg-1) than in that from SmT (467 nmol mg-1). There was also a clear distinction in Man content between fractions A of SmO (113 nmol mg-1) and SmT (732 nmol mg-1). Glc (SmO, 697 nmol mg-1; SmT, 616 nmol mg-1) and Gal (SmO, 113 nmol mg-1; SmT, 204 nmol mg-1) were also present in both samples. For linkage analysis, fraction A was methylated (34), hydrolyzed, reduced, and acetylated. GC/MS analyses of the resulting partially methylated alditol acetates identified two terminal, two 4-substituted, one 3-substituted, one 6-substituted, and one 3,4-disubstituted hexoses. Pentose (arabinose) residues were present as terminal, 5-substituted, 2-substituted, and 3,5-disubstituted furanoses. Another methylation analysis involving MeI-D3 revealed that 6-O-methyl-hexose was 4-substituted, whereas the 3-O-methyl-hexose occurred as both a terminal and a 4-substituted residue. In SmO the presence of 4-substituted Hexp was more than twice as abundant as in SmT (SmO, ~20%; SmT, ~9%). Compared with fraction A of SmT, only one-third of the 6-substituted Hexp was present in SmO (SmT, ~12%; SmO, ~4%).

Fractions B consisted exclusively of Ara and Man and thus were pure AMs. A distinct difference in the Ara/Man ratio was identified between the AMs obtained from SmO and from SmT. In the AMs of SmO, an approximate molar ratio of Ara/Man of about 1:1 was identified (2070 nmol mg-1 Ara; 2201 nmol mg-1 Man), whereas in the AMs of SmT a ratio of ~3:10 (692 nmol mg-1 Ara; 2319 nmol mg-1 Man) was found. Methylation analyses of both AMs revealed the presence of the same substituted sugars but in different ratios (data can be found in the supplemental information, Table S1). In both AMs four differently substituted Araf and six differently substituted Manp residues were identified. However, there was a clear structural difference between these polysaccharides. In agreement with the results of neutral sugar analyses, a total of 60% hexose residues was identified in the AMs from SmT and only 41% in the AMs from SmO. With regard to the pentose residues, the polysaccharides differed in the content of both the 5- and 2-substituted Araf residues. The AMs from SmT contained ~18% 5-substituted Araf and the AMs from SmO 28%. The quantity of 2-substituted Araf was less in SmT (~7%) than in SmO (~12%). Regarding hexose residues there was a clear difference in the amount of 6-substituted Manp. The content of this residue in the AMs from SmT (19%) exceeded that in the AMs from SmO (6%). Branched 2,6-disubstituted Manp residues were twice as abundant in the AMs from SmT (~12%) than those from SmO (6-7%). 2-Substituted Manp (~6% in SmT and SmO) as well as 3-substituted Manp (5% in SmT, ~4% in SmO) occurred in both AMs in similar content. t-Manp residues were also present in similar concentrations (SmT, 15%; SmO, 17%). Thus, the results of methylation analyses were consistent with structural differences between both AMs as indicated by compositional analyses. The polysaccharide from SmT possessed a larger mannan (~60%) and shorter arabinan (~40%), and the AMs from SmO comprised a truncated mannan (41%) and a larger arabinan (~59%).


Figure 2
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  		FIGURE 2.
Expanded regions (4.85-5.35 ppm) of one-dimensional 1H-NMR spectra in D2O at 300 K of capsular AM from M. avium 2151 SmT (a) and SmO (b).

 
NMR Spectroscopy—NMR experiments identified a pyranosidic ring form for all hexoses and a furanosidic configuration for all pentose/arabinose residues. Signals at 0.9 and 1.3 ppm demonstrated the presence of fatty acids in fraction A, showing that at least some molecules were acylated. Thus, chemical and NMR spectroscopy analyses revealed that fractions A contained lipoglycans, probably including LAM. These structures were not investigated further.

One-dimensional 1H-NMR spectra of the AMs from SmT and SmO (Fig. 2) showed at least 10 partially overlapping anomeric signals (A-J, 5.5-4.9 ppm) with chemical shifts typical for the {alpha}-configuration. The presence of fatty acids could be excluded because of missing signals around 0.9 ppm (-CH3) and 1.3 ppm (-CH2-). In both spectra very similar chemical shifts, but with different intensities, were identified. Two-dimensional NMR experiments, namely COSY and HMQC (supporting information in supplemental Figs. S1 and S2, respectively), allowed overlapping signals to be distinguished. In the case of the AMs of SmT, the resonance at 5.13 ppm (Fig. 2a) comprised three anomeric signals. One of these (at 5.123 ppm) correlated with an H-2 resonance at 4.300 ppm (G1,2 in supplemental Fig. S1a), another one at 5.123 ppm with an H-2 resonance at 4.042 ppm (F-c1,2), and a third at 5.132 ppm was correlated to an H-2 resonance at 4.034 ppm (F-b1,2). Furthermore, two overlapping signals around 5.09 ppm were distinguished with chemical shifts at 5.089 and 5.098 ppm (H-b1,2 and H-a1,2, respectively). Also, the most abundant resonance at 5.045 ppm (I in Fig. 2) comprised two signals at 5.031 and 5.048 ppm (I-b1,2 and I-a1,2, respectively, in supplemental Fig. S1a).

Likewise, overlapping signals in the 1H-NMR spectrum of the AMs from SmO could be differentiated. The signal at 5.12 ppm (Fig. 2b) comprised two resonances at 5.116 and 5.132 ppm (G1,2 and F1,2, respectively, in supplemental Fig. S1b). Furthermore, the signals H and C (at 5.09 and 5.17 ppm, respectively) could be differentiated between H-a, H-b, and H-c and between C-a and C-b.

Proton chemical shifts were assigned by COSY and TOCSY spectra and carbon chemical shifts by HMQC and HMBC spectra. In both AMs, all sugar residues possessed the {alpha}-configuration. For all Man residues, the {alpha}-configuration was assigned in HMQC experiments without decoupling (spectra not shown) by determination of the coupling constants (JH1,C1 > 170 Hz). The identification of {alpha}-configured Ara residues was possible because of the chemical shifts of the respective C-1 atoms. In the AMs from SmT, six different Ara residues were identified (A, B, C, G, H-a, and H-b, in supplemental Table S2), all of which were furanoses. Chemical shifts of single 13C atoms were compared with those of unsubstituted monosaccharides (35) and resulted in the identification of substituted carbon atoms. The C-2 resonances of Ara residues A and B were shifted downfield (~{Delta} 2.4 ppm), thus proving that these residues were substituted at position 2. In residue G the C-3 resonance was shifted downfield (~{Delta} 2.4 ppm), identifying this sugar as 3-substituted Ara. Whether this residue was a 3,5-disubstituted Ara, which was detected in methylation analysis (~16 mol %, supplemental Table S1), could not be clarified. The C-5 resonances of Ara residues H-a and H-b were shifted downfield (~{Delta} 2.2 ppm) and therefore identified a substitution at O-5. Eight different Man residues were identified (D, E, F-a, F-b, F-c, I-a, I-b, and J, supplemental Table S2), all of which were pyranoses. Chemical shifts of the C-2 of Man residues D and E proved their substitutions at O-2 ({Delta} 3.6 ppm for C-2 of D and {Delta} 2.9 ppm for C-2 of E) (36). According to methylation analysis, which identified 2,6-disubstituted in addition to 2-substituted Man residues, for Man D and Man E an additional substitution at O-6 could be possible. However, because of strong overlapping, such substitution could not be confirmed by NMR spectroscopy. On the contrary, Man F-a, Man F-b, and Man F-c were clearly identified as 2,6-linked Man residues because of downfield shifts of each C-2 ({Delta} 8.6-8.7 ppm) and C-6 ({Delta} 4.6-5.6 ppm). Resonances of C-2, C-3, C-4, and C-6 of Man I-a and Man I-b were not shifted at all (35); therefore, these were terminal residues. C-6 of Man J was shifted downfield ({Delta} 1.3 ppm) identifying it as 6-substituted Man.

In the same way substitutions were assigned for sugar residues of the AMs from SmO (supplemental Table S3). The sugar sequences of the AMs were identified by ROESY experiments. In the spectrum of the AMs from SmT (supplemental Fig. S3a) both protons D1 and E1 showed NOE connectivities to proton B2. This demonstrated 2-substituted Manp residues being linked to position 2 of Araf residues. Furthermore, interresidual effects between proton I-a1 and proton F-c2 were observed showing t-Manp linked to position 2 of 2,6-disubstituted Manp. An NOE contact between proton F-a1 and proton J6 demonstrated the substitution of 6-substituted Manp with 2,6-Manp. Additionally, an interresidual contact between proton J1 and proton F6 gave evidence for a 1 -> 6 chain built up of 6-substituted and 2,6-disubstituted Manp, the last of which is substituted at position O-2 with t-Manp. The linkage of t-Manp to position O-2 of 2-substituted Manp was identified by an NOE contact between proton I-b1 and proton D2.


Figure 3
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  		FIGURE 3.
Charge-deconvoluted positive ion mode ESI FT-ICR mass spectrum of the AM from SmO. The spectrum was recorded in the positive ion mode, and afterward the charge was deconvoluted. The capillary exit voltage was set to 250 V. {Delta} and *, indicate mass differences of 132 and 162 atomic mass units, respectively. The inset shows a detail of the mass region of the most abundant molecule. {Delta}m =±30 atomic mass units, indicating the mass difference resulting from the exchange of the hexose by a pentose.

 
In the ROESY spectrum of the AMs from SmO (supplemental Fig. S3b), interresidual contacts were observed between proton B1 and proton G3, demonstrating the linkage of 2-substituted Araf to position 3 of 3-substituted Araf. The NOE connectivity between proton D1 to proton B2 proved the 1 -> 2 linkage between 2-substituted Araf and 2-substituted Manp. An interresidual contact between proton G1 and proton C5 demonstrates that 5-substituted Araf was linked to position 5 to 3-substituted Araf. NOEs between proton J1 and E6 and between I-b1 and F2 show 6-Manp linked at position 6 and t-Manp linked at position 2 of 2,6-Manp.

Mass Spectrometry—To determine the molecular masses of the AMs, mass spectrometry was performed. The spectra comprised a complex of molecules each in different charge state. To simplify the interpretation, the mass spectra were charge-deconvoluted leading to the presence of only neutral species. Fig. 3 depicts the deconvoluted ESI mass spectrum of the AMs from SmO. Numerous signals reflected the heterogeneity of the polysaccharide and the fragmentation induced by capillary skimmer dissociation. The spectrum comprised a broad distribution of ions between 700 and 4500 atomic mass units, with characteristic mass differences of {Delta}m = 22, 30, 132, and 162 atomic mass units. Mass differences of {Delta}m = 22 atomic mass units were due to additional sodium adducts. Mass differences of {Delta}m = 162 atomic mass units corresponded to the mass of one hexose residue. Appropriately, differences of {Delta}m = 132 atomic mass units were due to pentose residues. Based on the results of neutral sugar analyses, hexose was Man and pentose was Ara. {Delta}m = 30 atomic mass units resulted from the "exchange" of one Ara for one Man residue. There were no further mass differences, consistent with the presence of different monosaccharides or contaminations. Each signal represented an ion of the polysaccharide, comprising Ara and Man residues only. Based on the neutral sugar analyses, mass peaks could be explained by different numbers of Ara and Man residues. Combinations were calculated and compared with the measured mass peaks, and measured and calculated masses were in excellent agreement (supplemental Table S4). It was obvious that all abundant signals represented fragments comprising more Ara than Man. In each fragment Ara was at least twice as abundant as Man. The most abundant peak was detected at 2558.791 atomic mass units representing the mass of a molecule comprising 14 Ara and 4 Man residues (+2 sodium ions). The expanded region (Fig. 3) shows completely separated isotope distributions including lower abundant signals, which can also be explained as sodium adducts consisting of 15 Ara and 3 Man (2528.785 atomic mass units) as well as 13 Ara and 5 Man (2558.804 atomic mass units) residues.

The deconvoluted ESI mass spectrum of the AMs from SmT (supplemental Fig. S4 and Table S5) also showed numerous signals between 1100 and 4600 atomic mass units, with characteristic differences of 22, 30, 132, and 162 atomic mass units, which were caused by masses of sodium, Ara, and Man, respectively. One single fragment consisted exclusively of Man (fragment 2 in supplemental Table S5). Such a fragment was not detected in the AMs from SmO. Furthermore, in comparison with the AMs from SmO, this spectrum showed fragments comprising more Man than Ara residues (fragments 2, and 8-11 in supplemental Table S5); however, also one cluster of fragments with more Ara residues was present (fragments 1 and 3-6). This was in good agreement with the results of neutral sugar and methylation analyses (supplemental Table S1).

ESI MS analysis gave new information about structural features of the AM. The most abundant fragments of both AMs consisted of Ara and Man. There are hardly any fragments comprising only Ara or only Man. Few signals (fragments 3, 7, and 15 in supplemental Table S4 and 1, 3, and 6 in supplemental Table S5) were present in both AMs representing fragments with Ara residues dominating. The AMs from SmT showed fragments with a maximum of 20 Man residues (fragment 11, supplemental Table S5), whereas in the spectrum of the AMs from SmO only fragments with not more than seven Man residues were found (fragments 15 and 16, supplemental Table S4). Compared with the AMs from SmO, the number of Man residues was significantly higher in the AMs from SmT, which was consistent with the data from chemical analyses. Based on these data, structural models of the AMs of M. avium 2151 SmO and SmT are proposed (Fig. 4).


Figure 4
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  		FIGURE 4.
Structural models of the AMs isolated from M. avium 2151 variant SmT (a) and M. avium 2151 variant SmO (b). One possible structure of each AM is depicted. A, arabinose; M, mannose.

 
Induction of Cytokines—To define the biological activity of the M. avium-derived AMs and acylated polysaccharides, we used in vitro stimulation assays of murine and human immune cells. Stimulation of murine bone marrow-derived macrophages as well as human DC with lipoglycan fractions (fraction A in Fig. 1) and AMs (fraction B in Fig. 1) from SmO and SmT resulted in the production of both immunostimulatory cytokines (TNF-{alpha}/IL-12) and the immunoregulatory cytokine IL-10 (Fig. 5; other data can be found in supplemental Fig. S5). TNF-{alpha} and IL-10 levels were comparable with those induced by LPS used as positive control. In contrast, AMs only slightly induced cytokine release even after stimulation with a high dose of 10 µg/ml. All fractions were tested for endotoxin contaminations via a LAL (Limulus amebocyte lysate) assay. Lipoglycan fractions contained LPS in concentrations of 100 pg µg-1 sample (SmT) and 81 pg µg-1 (SmO). In AMs only marginal endotoxin concentrations of 15 pg µg-1 sample (SmT) and 3 pg µg-1 (SmO) were detected.


Figure 5
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  		FIGURE 5.
Cytokine release of human dendritic cells after stimulation with acylated polysaccharide fractions (PS) and AMs from SmO and SmT. a, TNF-{alpha}; b, IL-10; c, IL-12. Human dendritic cells (106 ml-1) were cultivated with the stimuli (10 µgml-1) at 37 °C in 5% CO2. After 18 h the concentrations of TNF-{alpha}, IL-10, and IL-12 in the supernatants were measured by enzyme-linked immunosorbent assay. Ctrl., control (immature dendritic cells); LPS, lipopolysaccharide from Salmonella enterica sv. Minnesota, strain R345 (10 ng ml-1).

 


Figure 6
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  		FIGURE 6.
Cytotoxicity of human MNC after stimulation with acylated polysaccharide fractions (PS) and AMs from SmO and SmT against the human bladder carcinoma cell line T24. MNC (106 ml-1) were cultivated with the stimuli (10 µgml-1) for 7 days at 37 °C in 5%CO2. 51Cr release of labeled tumor cells was measured after 5 h of cocultivation with stimulated effector cells. Specific lysis was determined according to the formula "specific lysis (%) = (Reff - Rspo)/(Rmax - Rspo) x 100" where Reff is experimental release, Rspo spontaneous release, and Rmax maximum release. Effector/target ratios were 10:1, 20:1, and 40:1. Ctrl., control (unstimulated cells); BAK, BCG-activated killer cells.

 
In summary, the cytokine measurements revealed that the induction of proinflammatory TNF-{alpha} as well as IL-12 and antiinflammatory IL-10 depends on structural features, namely acylation of the stimulus. This was shown in murine macrophages as well as in human dendritic cells.

Anti-tumor Cytotoxicity—Because of their immunomodulatory properties, mycobacterial AMs are used in vaccination and immunologic cancer therapy. Therefore, to further assess the biological activity of the lipoglycan fractions (fraction A in Fig. 1), and AMs (fraction B in Fig. 1), human MNC were stimulated and tested for cytotoxicity against the human urinary bladder carcinoma cell line T24. After 5 h of cocultivation of effector and 51Cr-labeled target cells, the released radioactivity was measured, and specific lysis was calculated. As depicted in Fig. 6 both AMs and lipoglycan induced substantial anti-tumor cytotoxicity of human MNC. All tested fractions reached between 40 and 60% of the activity achieved with BCG, which has been described as a potent stimulator. No significant difference between AMs and lipoglycan or between SmO and SmT was observed. Interestingly, this indicates that activation of human cytotoxic effector cells, unlike cytokine release of dendritic cells, is independent of the lipid domain.

Repeated experiments revealed that stimulation with AMs and lipoglycan fractions does not induce apoptosis of monocytes (data not shown). An apoptosis-inducing effect can be excluded for concentrations of 10 µgml-1 stimulus.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The mycobacterial cell envelope represents a complex and unique structure compared with cell envelopes of other prokaryotes, as it is composed mainly of polysaccharides, lipoglycans, proteins, and peptidoglycan. In addition, the cell envelope of some mycobacterial species is surrounded by an outermost layer described as a capsule. Constituents of this capsule represent potential virulence factors because they could be involved in the first steps of host cell infection or in cell protection of intraphagosomal mycobacteria. M. avium, a facultative intracellular opportunistic pathogen in humans, is a well studied model for mycobacterial infections. A major phenotypic characteristic of M. avium isolates is their ability to appear in different colony morphotypes: smooth (either transparent or opaque) or rough (reviewed in Ref. 20). Although morphotypes of the same strain are very closely related, previous studies have demonstrated that the smooth-transparent variant is usually better able to survive and grow intracellularly than the opaque morphotype (38).

To elucidate whether structural differences in the mycobacterial capsule composition may explain the different biological activities of these isolates, the structural characteristics of capsular polysaccharides from M. avium strain 2151 SmT and SmO were determined. Neutral AMs were separated from a lipoglycan fraction, most likely comprising LAM. It is noteworthy that this fraction was recovered after a mild extraction technique using 1% phenol. Usually for extraction of lipoglycans the French press method is used. Our finding supports the hypothesis that LAM is not only membrane-associated (37) but also is located in the outermost layer of the mycobacterial cell wall as first proposed by Rastogi (38).

Analysis of the AMs revealed structural differences between SmT and SmO. The AMs from SmT possessed a relative molar ratio of Ara:Man of 1:4, whereas that from SmO contained Ara and Man in similar proportions. Substitution patterns of Ara and Man were in accordance with published structures of other mycobacterial AMs (39). Interestingly, the AMs isolated herein showed lower molecular masses compared with that described for M. tuberculosis. A further structural characterization was achieved by various NMR experiments; however, the occurrence of a number of overlapping signals did not allow a full assignment of all protons. In both AMs, the NMR data proved a (1 -> 6)-linked mannan main chain with alternating 6-substituted and 2,6-disubstituted Man residues, the latter being substituted at position 2 by a terminal Manp. A (1 -> 5)-linked arabinan backbone was substituted in position 3 with a (1 -> 5)-linked side chains. It is not clear whether 5-substituted and 3,5-disubstituted Araf residues were alternating or whether (1 -> 5)-linked chains were located between the branching points. The length and position of the side chains could not be determined. t-Manp residues represented "cap" structures of the side chains, directly substituting (1 -> 5)-linked Araf and branching from (1 -> 2)-linked Manp. Probably, also cap structures composed of (1 -> 3)-linked Manp residues were present. The linkage of the arabinan to the mannan domain remained unidentified.

Mass spectrometric analyses of the isolated AMs gave further information about their structural features. Based on chemical and NMR data, particular fragments in ESI FT-ICR mass spectra could be assigned as side chains. Fragments composed exclusively of Manp residues were identified only in the case of the AMs from SmT. A reason for this could be that this mannan showed a different fragmentation behavior due to partial structures lacking branches, compared with the arabinan possessing larger side chains, i.e. such long-chain unbranched mannan fragments to a lower degree. For the arabinan possessing side chains, a stronger fragmentation was expected. Therefore, most of the identified fragments could be assigned as arabinan side chains (with more or less portions of the main chain). However, not every fragment represented a side chain. It had to be considered that each side chain of the postulated structure possesses an even number of mannose residues. The presence of signals corresponding to fragments with an odd number of mannose units could be explained by the loss of Man residues.

The AMs from SmO comprised a truncated mannan and a larger arabinan compared with the AMs from SmT. Because of the larger arabinan, a number of fragments could be explained in part as arabinan side chains (with or without units of the main chain). Further structural differences between the AMs from SmO and SmT could not be identified but were not excluded.

Aspects of the biological activities of all isolated AMs and lipoglycans were examined to determine the relevance of the lipid versus the sugar moieties and the possible differences between components of virulent SmT and nonvirulent SmO.

Lipoglycan fractions induced substantial amounts of proinflammatory TNF-{alpha} and immunoregulatory IL-10, whereas lipid-free AMs only marginally induced TNF-{alpha} production, which was not due to LPS contamination. Measurements of IL-10 production by murine bone marrow-derived macrophages after stimulation with lipoglycan fractions and AMs showed comparable results. After stimulation with the lipoglycan fractions, high concentrations of IL-10 were measured.

Because IL-12 is a key cytokine inducing strong TH1 immune responses and anti-tumor immunity, the capacity of our isolated components to induce production of bioactive IL-12 p70 in human DC was determined (40). Although production of bioactive IL-12 often requires co-stimulation with cytokine IFN-{gamma}, for example, lipoglycans but not AMs induced secretion of substantial amounts of IL-12 p70 in human DC.

The observation that a similar biologic activity was observed when human and murine macrophages were stimulated with acylated AMs isolated from both strains indicates that these structures are biologic response modifiers expressed by both morphotypes. However this also demonstrates that acylated AM are not responsible for the major differences with respect to macrophage activation observed in infection experiments with viable bacteria. The highly replicative smooth-transparent morphotype of M. avium strain 2151 induced significantly less phosphorylation of the mitogen-activated protein kinases p38 and ERK1/2 than the smooth-opaque morphotype of the same strain, which was gradually eliminated from macrophage cultures. These differences have also been seen on the level of cytokine formation (41, 42). To date, the molecular basis for these differential effects are not understood: A recent report by Bhatnagar and Schorey (43) indicates that the lack of glycopeptidolipids on the bacterial surface may correlate with an increased macrophage activation due to the presence of other macrophage-activating cell wall components.

Mycobacterial AMs have been described as potent biological response modifiers. For example, Z-100 (an AM-rich extract from M. tuberculosis) showed anti-metastatic activity in a murine B16 melanoma model (14). However, the preparations used in those studies also contained LAM, and thus the active compound remained unclear. Our studies found similar immunomodulatory activities of highly purified AMs from M. avium. We stimulated human MNC with both lipoglycan fractions and AM, and tested cells for activity against the human urinary bladder carcinoma cell line T24. Both lipoglycans and AMs induced profound anti-tumor cytotoxicity in the range of 40 to 60% of that obtained by M. bovis BCG. BCG has been described as inducing strong natural killer cell-mediated tumor cell lysis in this system and serving as a positive control (44, 45).

In conclusion, our experiments showed that both lipoglycan fractions and AMs have biological response modifying activity. However, a morphotype-specific difference was not detectable. Experiments with murine macrophages and human dendritic cells illustrated the importance of acylation for activation of myeloid immune cells. Cytokine induction by acylated mycobacterial AMs have been described (46-48). Gilleron et al. (49) showed that the lipid anchor in AraLAM from M. smegmatis is of key importance regarding cytokine induction. Other work supports the hypothesis that biological effects depend on the degree and chemical structure of capping motifs and particular substitutions on the arabinan moiety, i.e. succinate (50). Our investigations showed that neither in AMs nor in lipoglycan fractions obtained from M. avium morphotypes do such substitutions occur. Our data confirm that lipid moieties such as those present in LPS or bacterial lipoproteins are essential for cytokine induction.


   FOOTNOTES
 
* This work was supported in part by Deutsche Forschungsgemeinschaft Grant SFB 470 B1 (to O. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S5 and Tables S1-S5. Back

1 Present address: Dept. of Otorhinolaryngology, Head and Neck Surgery, University Hospital Essen, D-45122 Essen, Germany. Back

2 Present address: Dept. of Medicine, Division of Infectious Diseases, and Dept. of Molecular Virology, Immunology, and Medical Genetics, Center for Microbial Interface Biology, Ohio State University, Columbus, OH 43210. Back

3 Supported by Grant AI 018357 from NIAID, National Institutes of Health. Back

4 To whom correspondence should be addressed. Tel.: 49-4537-188472; Fax: 49-4537-188745; E-mail: oholst{at}fz-borstel.de.

5 F0The abbreviations used are: AM, arabinomannan; LAM, lipoarabinomannan; SmT, smooth-transparent; SmO, smooth-opaque; GC/MS, gas chromatography/mass spectrometry; COSY, correlation spectroscopy; TOCSY, total correlation spectroscopy; NOESY, nuclear Overhauser enhancement spectroscopy; ROESY, rotating frame Overhauser enhancement spectroscopy; HMQC, heteronuclear multiple quantum coherence; HMBC, heteronuclear multiple bond correlation; TNF, tumor necrosis factor; DC, dendritic cells; IL, interleukin; MNC, mononuclear cells; Araf, arabinofuranosyl unit; Manp, mannopyranosyl unit; t-Manp, terminal Manp; NOE, nuclear Overhauser effect; ConA, concanavalin A. Back


   ACKNOWLEDGMENTS
 
We thank Zbigniew Kaczynski for recording the NMR spectra and Regina Engel, Svenja Kröger, and Gabriele Bentien for technical assistance.



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 DISCUSSION
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